Process for the temporary protection of amino groups in peptide syntheses

ABSTRACT

Temporary protection of amino groups by the group of the formula I   in which R1 represents lower alkyl, R2 lower alkyl or phenyl and R3 phenyl, and wherein the phenyl radicals are unsubstituted or substituted by lower alkyl, phenyl or lower alkylphenyl groups.

United States Patent 1 Iselin et al.

[ Apr. 1, 1975 PROCESS FOR THE TEMPORARY PROTECTION OF AMINO GROUPS INPEPTIDE SYNTHESES [75] lnventors: Beat Iselin, Riehen; Peter Sieber,

Reinach/Basel-Sand, both of Switzerland [73] Assignee: Ciba-GeigyCorporation, Ardsley,

22 Filed: Mar. 30, 1972 21 Appl. No.: 239,721

Related US. Application Data [63] Continuation-in-part of Ser. No.698,118, Jan. 16,

1969, abandoned.

[30] Foreign Application Priority Data Jan. 25, 1967 Switzerland 1073/67Aug. 28, 1967 Switzerland 12038/67 Nov. 6, 1967 Switzerland 17108/67[52] US. Cl 260/471 C, 260/1 12.5, 260/349,

[51] Int. Cl. C07c 103/52, C07c 117/00, C07c 103/20 OTHER PUBLICATIONSHiskey et al., J. Am. Chem. Soc., 87, 3969 (1965). Marshall et al.,Biochemistry, 4, 2394 (1965). Anderson et al., J. Am. Chem. Soc., 79,6180 (1957). Merrifield, J. Am. Chem.Soc., 86, 304 (1964). Sieber etal., Helv. Chem. Acta, 51, 614 (1968). Wolman, The Chemistry of theAmino Group," S. Patai, Ed., lnterscience Publishers, N.Y., 1968, pp.685,697.

Bailey et al., Polymer Preprints, 5, 266-281 (1964).

Primary ExaminerLewis Gotts Assistant Examiner-Reginald J. SuyatAttorney, Agent, or Firm.loseph G. Kolodny; John J. Maitner; Theodore O.Groeger [57] 1 ABSTRACT Temporary protection of amino groups by thegroup of the formula 1 OCO- (I) in which R represents lower alkyl, Rlower alkyl or phenyl and R phenyl, and wherein the phenyl radicals areunsubstituted or substituted by lower alkyl, phenyl or lower alkylphenylgroups.

18 Claims, No Drawings PROCESS FOR THE TEMPORARY PROTECTION OF AMINOGROUPS IN PEPTIDE SYNTHESES This is a continuation-in-part ofapplication Ser. No. 698,118 filed Jan. 16, 1968 now abandoned.

The present invention provides a new process for the temporaryprotection of amino'groups in peptide syntheses by acylating the aminogroup and then eliminating the acyl group introduced.

As is known is is in general necessary when synthesizing peptides bycoupling aminoacids or peptides to block any functional groups notparticipating in the coupling reaction, for example the terminal aminogroup or the terminal carboxyl group, the amino and carboxyl groups ofthe side-chains and possibly further groups such as mercapto, guanidinoor hydroxyl groups. While at this stage a considerable choice ofprotective groups are available, they are still not quite satisfactory,especially so when long-chain and delicate peptides are to besynthesized. The problem involved is above all that in such caseshydrogenolytic elimination is out of the question (because of sulfurousaminoacids present) and the peptides are not sufficiently stable towardshydrolytically acting acid or especially alkaline agents so that thehydrolytic elimination of all protective groups in the last stage of thesynthesis entails considerable losses of costly material.

It is another difficulty that the protective group located at thecoupling end must be eliminated again after every coupling reaction,whereas the other protective groups should be retained until thesynthesis is complete. These two types of protective groups musttherefore, on one hand, be selectively eliminable with respect to eachother and, on the other hand, both types must be eliminable under mildconditions.

In the peptide field, there exists therefore a need for protectivegroups which a. can be split off under mild conditions, preferably in aneutral or acid medium, so that peptide bonds of sensitive peptides arenot affected;

b. which can be split off without the use of catalysts when the peptidecontains sulfurous aminoacids, for example cystine;

c. which give good yields in each condensation method, for instancemixed anhydride method, solid phase method;

d. which can be split off with a selectivity of about 100 percent fromother protective groups required in the peptide synthesis;

e. which can easily be introduced into aminoacids or peptides.

No ideal protective groups for all kinds of peptides are available.Depending on the peptide to the synthesized, it is necessary to use adifferent combination of protective groups.

As side-chain protective groups eliminable under mild acidic conditionsthe tertiary butyloxycarbonyl group (BOC) has proved particularlysuitable for protecting the amino groups and the tertiary butyl estergroup (tBu) for protecting the carboxyl groups, and in addition thetertiary butyl ether group is available for protecting hydroxyl groups.Apart from these groups the trityl group for example when used as ana-amino protective group, can be selectively eliminated, since in aweakly acidic medium it is much faster eliminable, for example in 80percent acetic acid at room temperature 20,000 times faster.Unfortunately, however, the suitability of the trityl group as aminoprotective grouping is very limited. Owing to a steric hindrance itcannot be used in coupling according to the method of the activatedesters and of the mixed anhydrides (except in the case of glycine) andeven in the carbodiimide method it gives poor yields. Therefore, it isunsuitable for use in the synthesis of peptides starting from thecarboxyl end, for example by the new method of the solid phase synthesis(of. Merrifield, J. AM. Chem. Soc. 85, 2149 [1963]). The trityl grouphas also other disadvantages because it is difficult to introduce andcompounds protected by the trityl group are not very stable.

The present invention is based on the observation that protective groupsof the formula O-CO- where R is lower alkyl, R; lower alkyl or phenyland R represents phenyl can be used with advantage in the synthesisoflong-chain, delicate peptides, in fact with better results than knownprotective groups, for example the trityl group. The phenyl radicals inthe above formula represent an unsubstituted phenyl ring or a phenylring substituted by one or two lower alkyl, phenyl or lower alkylphenylgroups. The substitucnts are above all in para-position, though they mayalso be in ortho-position or in the orthoand parapositions. The loweralkyls contain at most 5 carbon atoms and are in the first place methylor ethyl or, for example, propyl or butyl radicals. The lower alkylgroups are linear, though especially in the case of the phenylsubstituents they may also be branched.

The new groups are distinguished by the fact that they are eliminableunder very mild acidic conditions, for example at room temperature inabout 60 to percent aqueous acetic, chloracetic or formic acid or in amixture of at least two of these acids and 10 to 40 percent of water.Since the speed at which they are eliminated is at least 600 timesgreater than that of the BOC group, they can be eliminated selectivelywith respect to this group and also to the tertiary butyl ester group,the tertiary butyl ether group and the trityl-mercapto protective group.They are therefore particularly suitable as a-amino protective groups inthe synthesis of delicate peptides containing acid-labile sidechainprotective groups, such as the groups just mentioned. Since they displayno signs of a steric hindrance, they can be used in any desired couplingmethod and especially also in the solid phase synthesis.

An object, of the invention is therefore a process for the temporaryprotection of amino groups in peptide syntheses by acylating the aminogroup to be protected before carrying out the coupling step(s) by aprotective group of the formula I in which R represents lower alkyl, Rlower alkyl or phenyl, and R stands for phenyl, and wherein the phenylradicals are unsubstituted or substituted by lower alkyl, phenyl orlower alkylphenyl groups and splitting off the protective group afterthe coupling step(s) has (have) been carried out.

An object of the invention are aminoacids and peptides and derivativesthereof which are protected by a group of the formula 1 as definedabove. A further object of the invention are means for introducing thenew acyl groups.

As mentioned above, the new protective groups are especially useful forprotecting aminoacids or peptides and derivatives thereof used inpeptide syntheses. By peptides are to be understood in the first linethose peptides which occur in nature and are, for instance, described inthe text book The Peptides by Schrbder and Liibke, Academic Press, NewYork and London, Volumes I and II, 1965-66, and also synthetic analoguesof such peptides which differ from them by having one or more aminoacidsexchanged by other aminoacids, those exchange aminoacids being known inthe peptide field. By peptides are further to be understood partialsequences of the natural or synthetic peptides just mentioned. Asaminoacids occurring in the peptides as building elements or structuralunits there may be mentioned the code aminoacids, cf. for instance Sci.American October 1960, pg. 55, and homologues, structural isomers andoptical isomers thereof, for instance amino-lower alkanoic acids with atmost 7 carbon atoms other than those occurring as code aminoacids, forinstance a-amino butyric acid, norvaline, norleucine, B-alanine,y-aminobutyric acid, a,B-diaminopropionic acid, further, for instance,hydroxyproline, normethionine, phenylglycine, ornithine, citrulline,N-methyl-tyrosine and other N-lower alkyl amino-acids, further racemicand D-aminoacids.

Derivatives of peptides are especially those which have substantiallythe same activity as the peptides themselves, for instance C-terminalamides, especially N-unsubstituted amides, but also N-monoor N-disubstituted amides, such as N-monoalkyl or N-dialkyl amides having upto 18, especially 1 to 5 carbon atoms in the alkyl group, or C-terminalesters, for instance alkyl esters having in the alkyl group up to 18,especially'l to 5 carbon atoms. Other derivatives are those which aregenerally used as intermediates in the synthesis of peptides, forinstance activated esters, hydrazides azides, mixed anhydrides andpeptides or derivatives in which, besides the amino group protectedaccording to the invention, one or more functional groups selected fromamino, carboxyl, hydroxy, mercapto, and guanidino groups are protectedin known manner by protective groups.

Such derivatives are also described in the abovementioned text-book ofSchrdder and Liibke or in publications of Wieland et a1.Peptidsynthesen, l to V, Angew. Chem. 63 (1951) 7-14; Le. 66 (1954),507-512; l.c. 69 (1957), 362-372; l.c. 71 (1959), 417-425, 1.0. 75(1963), 539-551. Derivatives especially to be mentioned are lower alkylesters for instance methyl ester, ethyl ester, tertiary butyl ester,tertiary amyl ester, benzyl ester, p-nitrobenzyl ester, pmethoxybenzylester, 2,2,2-trichloroethyl ester, 2- iodoethyl ester, p-nitrophenylester, 2,4-dinitrophenyl ester, 2,4,6- or 2,4,5-trichlorphenyl ester,2,3,4,5,6-pentachlorphenyl ester, N-hydroxysuccinimide ester, and otheractivated esters as mentioned for instance in US. Pat. No. 3,035,041;further C-terminal hydrazides and azides, further mixed anhydrides, forinstance anhydrides with carbonic acid lower alkyl ester such ascarbonic acid methyl ester or anhydrides with lower alkanoic acids whichmay be halogensubstituted, for instance with formic acid, pivaloic acid,trichloracetic acid; further derivatives of aminoacids of peptides inwhich one or more functional groups such as amino, carboxyl, hydroxy,mercapto and/or guanidino groups are protected.

It should be noted that the protective groups of the invention can beused in any method of peptide synthesis, and thaty they are alsoespecially useful in the solid phase method. They can be used inconnection with any protective groups, but it is of particular advantageto use them in'connection with protective groups which can be split offby means of acids, for instance the BOC group, the tert. butylestergroup and the tert. butyl ether group and analogous groups, because thenew groups have an excellent selectivity which respect to these knowngroups, which are preferably used in the synthesis of sensitivepeptides.

The new protective groups are introduced in known manner, similar to theBOC group, for example by way of the azide method or the method of theactivated esters (for example phenyl esters, p-nitrophenyl ester,hydroxysuccinimide ester) or by reaction of carbinols of the formula R RR C-OH with the isocyanic acid esters corresponding to the aminoacids.

The following Examples illustrate the introduction and elimination ofthe new group in the case of some natural a-aminoacids and of peptidessynthesized from natural a-aminoacids. In the same manner the groups areused with other amino-acids and peptides.

EXAMPLE 1 Introducing the 2-phenyl-isopropyloxycarbonyl group 1. Amixture of 5.35 g (41.5 mmols) of isocyanateacetic acid ethyl ester, 4ml of pyridine and 5.64 g (41.5 mmols) of phenyl-dimethylcarbinol isheated for 38 hours at 50C. The yellow solution is taken up in 60 ml ofether, at 0C agitated with molar citric acid and with water, dried oversodium sulfate and evaporated to dryness under vacuum. The residue istriturated with petroleum ether, the solid substance is filtered off andrefluxed in ml of hexane. The undissolved matter is filtered off, thefiltrate concentrated to half its volume, filtered through 0.5 g ofactive carbon and the N- (2phenyl-isopropyloxycarbonyl)-glycine ethylester is allowed to crystallize out; it melts at 69-70C. The thin-layerchromatogram of the product in chloroform reveals it to be unitary; Rf=0.4.

A solution of 675 mg (2.55 mmols) of the ester in 10 ml of dioxane ismixed with 1.47 ml of 1.9lN-NaOH and stirred for 1 /2 hours at roomtemperature. The clear solution is diluted with 20 ml of water, twiceextracted with ether, and the extract is washed twice with a smallquantity of water. The combined aqueous solutions are acidified at 0Cwith citric acid to pH 2, twice extracted with ether, the etherealextract is washed neutral, dried and filtered. Dicyclohexylamine is thenadded to the ethereal solution until an alkaline reaction has beenestablished, whereupon on cratching the salt begins to crystallize out.The ethereal solution is concentrated to about 10 ml, kept for a fewhours at 0C and allowed to crystallize, then the crystals of N-(2-phenylisopropyloxycarbonyl)-glycine-dicyclohexyl ammonium salt arefiltered off and washed with ether.

They melt at 162 to 163C with slight decomposition.

2. 60 Grams of phenyldimethylcarbinol (0.44 mol) in 450 ml of absolutemethylenechloride and 53 ml of absolute pyridine (0.66 mol) are mixed at-5C within 30 minutes with 67 ml of chlorocarbonic acid phenyl ester(0.53 mol) in 250 ml of absolute methylenechloride; a thick suspensionforms which is stirred on for 20 hours at C, then poured out over alittle ice and 100 ml of methylenechloride. The resultingmethylenechloride solution is repeatedly washed with water at 0C, driedover sodium sulfate and evaporated to dryness under vacuum at 20C. Theresulting, instable mixed carbonate is immediately dissolved in 180 mlof methanol, while being cooled with ice mixed with 52 ml ofhydrazinehydrate (1.06 mols) and kept at room temperature for another 20hours. The solution is then diluted with 600 ml of ether, washed withwater at 0C, then times with 0.5N-sodium hydroxide solution and withwater until the washings run neutral, dried over sodium sulfate andevaporated to dryness under vacuum at 50C. The residue (61 g) isdistilled under a high vacuum, to yield 52.1 g ofZ-phenyl-isopropyloxycarbonyl hydrazide in the form of a colorless,viscous oil which boils at 9598 under mm Hg pressure.

20.6 Grams of the above hydrazide (0.106 mol) are dissolved in 320 ml ofdimethylformamide, cooled to 20 to 30C and mixed with 160 ml of 1.94N-hydrochloric acid. while keeping the temperature below 20C, and then23.2 ml of 5-molar sodium nitrite solution are dropped in at to -10C.The whole is stirred on for 15 minutes at the same temperature and thenadjusted to pH=6 to 7 with saturated potassium carbonate solution. Themixture is extracted with 800 and with 400 ml of ether, the two etherealextracts are washed at 0C three times with water, combined, dried oversodium sulfate and evaporated to dryness under vacuum at 30C. Theresidue is cautiously distilled under a high vacuum and yields 17.5 g ofthe azide as a yellowish oil boiling at 475 1C under 0.005 mm Hg. Theinfrared spectrum contains the expected bands (azide band at 4.6 and47,11. [split up], carbonyl band at 5.8,u).

2.45 g (12 mmols) of 2-phenyl-isopropyloxycarbonylazide in 14 ml ofdimethylformamide are mixed with 5.75 g of N tertiarybutyloxycarbonyl-L-lysine methyl ester acetate (18 mmols) and 2 ml ofdimethylformamide, and in the course of 1 /2 hours at 0C 4.15 ml oftriethylamine are slowly stirred in dropwise. The clear solution is keptfor 3 days in a refrigerator, then diluted with 50 ml of ether and at 0Crepeatedly agitated with 0.1 molar citric acid solution and then withwater until the washings run neutral, dried over sodium sulfate andevaporated to dryness. The residue is dried under a high vacuum untilits weight remains constant and forms a chromatographically unitary,viscous oil. [a],, =+6i0.5 (c=2 in chloroform). Yield: 4.83 g =95percent of the theoretical of N (2-phenylisopropyloxycarbonyl)-Ntertiary butyloxycarbonyl-L-lysine methyl ester.

In the same manner as described under 1) theparadiphenyl-isopropyloxycarbonyl group can be introduced into theglycine ethyl ester, to yield the N-(2-pdiphenylisopropyloxycarbonyl)-glycine ethyl ester which crystallizesfrom methanol and melts at 122C.

EXAMPLE 2 Introducing the 2p-tolyl-isopropyloxycarbonyl group A mixtureof 6.22 g (41.5 mmols) of p-tolyl- 5 dimethylcarbinol, 4 ml of absolutepyridine and 5.35 g (41.5 mmols) of isocyanateaceticacid ethyl ester isheated for 38 hours at 50C and then worked up as described in Example 1,to yield 9.8 g of N-(2-p-tolylisopropyloxycarbonyl)-glycine ethyl esteras a yellow oil; Rf =0.250.35 in chloroform. For hydrolysis the ester isdissolved in 100 ml of 80 percent methanol and on the pH meter rapidlymixed with 24 ml of 1.9N- sodium hydroxide solution, whereupon the pHrises to 11.8. After 7 minutes, ZN-hydrochloric acid is added until pH8.5 is reached. The resulting solution is concentrated under vacuum toabout half its volume, 50 ml of water are added and the whole isextracted twice with ether. The ether solutions are washed twice withwater, the combined aqueous solutions acidified at 0C with citric acidto pH=2 and extracted with ether. The ethereal extract is washedneutral, dried over sodium sulfate, filtered, alkalinized withdicyclohexylamine, concentrated to about 30 ml, mixed with an equalvolume of petroleum ether and allowed to crystallize, to yield 8.4 g ofthe N-(2p-tolylisopropyloxycarbonyl)- glycine dicyclohexyl ammonium saltmelting at l45-l47 and, after recrystallization from ethyl acetate, at147-148C with decomposition.

EXAMPLE 3 Eliminating the 2-phenyl-isopropyloxycarbonyl group 1. 418.6mg of N-(2phenyl-isopropyloxycarbonyl)- glycine dicyclohexyl ammoniumsalt are dissolved at room temperature in 4.2 ml of a mixture of 7 partsby volume of glacial acetic acid, 1 part by volume of 82.8 percentformic acid and 2 parts by volume of water. After 4 hours thin-layerchromatographic analysis reveals that about 95 percent of the protectinggroups have been eliminated. After standing for 6 hours at roomtemperature, 20 ml of acetone are added, the precipitated glycine isfiltered off and thoroughly washed with acetone; it gives a clearaqueous solution and is chromatographically unitary. Yield: 68.5 mg=9 l.4 percent of theory. Under identical conditions tertiarybutyloxycarbonyl-glycine ethyl ester remains unchanged.

2. 71.7 mg (0.27mmol) of N-(Z-phenyl-isopropyloxycarbonyl)-glycine ethylester are dissolved at 25C in 1.35 ml of 80 percent acetic acid. Afterdifferent intervals specimens of 0.2 ml each are taken, introduced into4 ml of dimethylformamide and titrated with 0.1N- perchloric acid inglacial acetic acid (to determine the quantity of liberated glycineethyl ester). The half-time value for the elimination of the protectivegroup is about 2 hours. After 24 hours quantitative elimination has beenachieved, and even in the thin-layer chromatogram no trace of startingmaterial can be detected. Under identical conditions the N-protectivegroup of N-tertiary butyloxycarbonyl-glycineethyl ester remainsunchanged.

In an identical manner N-(2p-diphenyl-isopropyloxycarbonyl)-glycineethyl ester is split quantitatively within 3 /2 hours.

3. 68.8 mg of N-(2phenyl-isopropyloxycarbonyl)- glycine ethyl ester aredissolved at room temperature in 2.6 ml of 60percent chloracetic acid.After different intervals specimens of 0.20 ml each are taken, addedQuantitative splitting after Hydrolyzing medium 60% acetic acid 12 hours90% acetic acid 50 hours 75% formic acid immediately methanol+82.87(formic acid 1 1 11 hours glacial acetic acid-182.8% formic acid water 7l 2 4V2 hours glacial acetic acid 82.8% formic acid water 7 l 2 sodiumchloride 40 minutes 4. A solution of 439.7 mg (1.04 mmols) of N -(2-phenyl-isopropyloxycarbonyl)-N -tertiary butyloxycarbonyl L-lysinemethyl ester in 4.4 ml of 80 percent acetic acid is kept for 48 hours atroom temperature. Thin-layer chromatography after this time reveals thatthe N -protective group has been eliminated practically quantitativelybut the N -tertiary butyloxycarbonyl group has not been affected. Thesolution is evaporated to dryness under vacuum, and the residue is driedunder a high vacuum at 45C, then dissolved in a small quantity of ether,whereupon slow crystallization sets in; to complete this, anapproximately equal quantity of petroleum ether is added, the whole isleft to itself for a few hours at C and then filtered, to furnish 269 mgof crystalline N -tertiary butyloxycarbonyl-L-lysine methyl esteracetate, melting at 808lC. The mother liquor further contains some N-tertiary butyloxycarbonyl-lysine methyl ester.

EXAMPLE 4 Eliminating the 2-p-tolyl-isopropyloxycarbonyl group 1. 432.6mg (1 mmol) of N-(2-p-tolyl-isopropyloxycarbonyl)-glycine dicyclohexylammonium salt are dissolved in 4.3 ml of a mixture of 7 parts by volumeof glacial acetic acid, 1 part by volume of 82.8 percent formic acid and2 parts by volume of water and stirred at room temperature. Afterminutes and after 30 minutes a thin-layer chromatographic analysis eachis carried out which reveals that after 15 minutes the splitting isabout 95 percent and after 30 minutes quantitative. Likewise after 30minutes, the solution is mixed with ml of acetone, allowed tocrystallize for a short time at 0C, and the glycine is then filtered offand thoroughly washed with acetone. Yield: 69.5 mg=92.7 percent ofcrystalline glycine.

2. When N-(2-p-tolyl-isopropyloxycarbonyl)-glycine ethyl ester istreated as described in Example 3 (2) for the corresponding2-phenyl-isopropyloxycarbonyl compound with 80 percent acetic acid at C,the protective group is split off quantitatively within 1 /2 hours.

EXAMPLE 5 Introducing the 2-p-diphenyl-isopropyloxycarbonyl group a.With the azide in aminoacid ester 106 Grams (134 0.5 mol) ofp-diphenyldimethylcarbinol in 500 ml of methylenechloride and 60 ml ofpyridine are mixed within minutes at -5C with a solution of 76 ml ofchlorocarbonic acid phenyl ester in 250 ml of methylenechloride; theresulting suspension is stirred for 14 hours at 0C, whereupon an almostclear solution is obtained which is evaporated to dryness under vacuumat 30C in a rotary evaporator. The crystalline residue, consisting of2-(p-diphenyl)- isopropyloxycarbonyl-phenyl ester, is dried for 1 hourunder a high vacuum at room temperature, then mixed with 200 ml ofdimethylformamide and 125 ml of hy- 0 drazine-hydrate and while coolingwith cold water the whole is stirred for 6 hours. The clear solution isslowly mixed with 1 liter of water while being cooled with ice, allowedto crystallize overnight at 0C, and the crystals are then filtered offand washed with N-sodium hydroxide solution and water and dried undervacuum at 50 C. The resulting 2-(p-diphenyl)-isopropyloxycarbonylhydrazide is recrystallized from 200 ml of carbon tetrachloride and 40ml of petroleum ether. Yield: 103.0 g (=76 percent of theory); meltingpoint l08-109C.

While stirring a solution of 27 g (0.1 mol) of this hydrazide in 270 mlof acetonitrile at -25C it is mixed with a solution of 50 ml of6N-hydrochloric acid in 100 ml of acetonitrile and then with 22 ml of5-molar sodium nitrite solution. After stirring the batch for 15 minutesat -l5C it is adjusted with 2N-soda solution to pH 6 to 7 and thesolution is poured into much ice water. After stirring for a short time,the azide solidifies; it is filtered off and washed with ice water. Theresidue is dissolved in ether, the water separated, the etherealsolution dried over sodium sulfate and evaporated at room temperatureunder vacuum, to yield 28.3 g (=lO0 percent of theory) of the azide inthe form of a crystalline yellowish powder melting at 48-52C.

While stirring 3.37 g (20 mmols) of L-valinemethylester hydrochloride in10ml of dimethylformamide at 0C there are added 2.8 ml of triethylamine,6.2 g of the above azide and 5 ml of dimethylformamide. After 10 minutesanother 2.8 ml of triethylamine are added and the whole is stirred onovernight at room temperature. The reaction mixture is diluted with 100ml of ether, at 0C repeatedly extracted with 0.1 molar citric acidsolution and water, dried over sodium sulfate and evaporated to drynessunder vacuum. The resulting ester is hydrolyzed as it is obtained. Forthis purpose the crude product is dissolved in ml of isopropanol, mixedwith 12 ml of 2N-sodium hydroxide solution, and the whole is stirred for2 /2 hours at 40C. Ether and water are then added to the solution, theaqueous solution is separated and mixed once more with water. Thecombined aqueous solutions are covered with ether, adjusted at 0C withmolar citric acid to pH=2 and extracted with ether. The etherealsolution is washed neutral and dried, and then rendered alkaline withcyclohexylamine, whereupon the cyclohexyl ammonium salt crystallizesout. The ethereal solution is concentrated to about 50 ml; mixed with 50ml of petroleum ether and allowed to crystallize at 0C. The crystallineproduct is filtered off and washed with ether+petroleum ether (1:1), toyield 6.5 g of N-2-(pdiphenyl)-isopropyloxycarbonyl-L-valine cyclohexylammonium salt melting at 178-l80C with decomposi'tion.

b. With the azide in amino acids: 1.17 g (10 mmols) of L-valine aredissolved with heating in 9.1 g of a 10 percent aqueous solution oftetramethylammonium hydroxide. The solution is evaporated under vacuumat C, and the residue is taken up three times in 5 ml ofL-proline-dicyclohexylammonium salt dimethylformamide and evaporatedunder vacuum. The crystalline residue is stirred with 20 ml ofdimethylformamide at room temperature, mixed with 2.8 g ofZ-p-diphenyloxycarbonyl azide, then with 3 ml of triethylamine and thesuspension is stirred for 1 hour at room temperature, then mixed withether and water, and the aqueous solution is separated, the etherealsolution extracted twice more with a little water and the combinedaqueous solutions are acidified at C to pH=2. The aqueous solutions areextracted with ether, the ethereal extracts washed with neutral, driedover sodium sulfate, filtered an rendered alkaline with cyclohexylamine.The cyclohexyl ammonium salt begins to crystallize immediately and isisolated as described under (a), to furnish 2.96 g of N- 2-(p-diphenyl)-isopropyloxycarbonyl-L-valine-cyclohexyl 7:1:2 ammonium salt melting atl78180C with decomposition.

0. With activated ester in aminoacids 1.17 Grams (10 mmols) of L-valineare dissolved in 4.55 ml of a 2.2N-methanolic solution ofbenzyltrimethylammonium hydroxide, evaporated to dryness under vacuum,once more dissolved in 10 ml of dimethylformamide and again evaporatedunder vacuum. The residue is taken up in 5 ml of dimethylformamide,mixed with 4.0 g of Z-(p-diphenyl)-isopropyloxycarbonyl phenyl ester andthe whole is stirred for 4 hours at 50C. The clear solution is mixedwith ml of water and 30 ml of ether and agitated; the aqueous phase isseparated and adjusted at 0C to pH=2 with citric acid and extracted withether. The ethereal extract is washed neutral, dried, filtered and mixedwith 1.9 ml of cyclohexylamine, whereupon the cyclohexyl ammonium saltof N-Z-(p-diphenyl)-isopropyloxycarbonyl-L- valine begins to crystallizeimmediately and is isolated as described under (a). The yield amounts to3.15 g. Melting point 178-180C with decomposition.

In an identical manner the followingN-2-(pdiphenyl)-isopropyloxycarbonyl derivatives can be prepared:

Derivative melting point 192 193C (decomp.) 227 230C (decomp.) l73 175C(decomp.)

Glycine-dicyclohexylammonium salt L-leucine N -tertiarybutyloxycarbonyl-L-lysine-dicyclohexylammonium saltL-tyrosine-cyclohexylammonium salt amorphous 248 252C (decomp.)

sinters at 158C L-phenylalanine-dicyclohexylammonium s t 116 119C(decomp.) L-glutamic acid-y-tertiary butylestercyclohexylammonium saltL-serine-tertiary butylether-cyclohexylammonium saltL-methionine-dicyclohexylammonium salt 174 175C (decomp.)

180 181C (decomp.)

2-(p-Diphenyl)-isopropyloxycarbonyl phenyl ester is prepared in thefollowing manner:

21.2 Grams of p-diphenyl-dimethylcarbinol (0.1 mol) in 100 ml ofmelthylenechloride and 12 ml of pyridine are mixed at 5C within 30minutes with a solution of 18.8 g of chlorocarbonic acid phenyl ester in50 ml of methylenechloride.- The batch is then stirred for 18 hours at0C, poured over ice water, the organic phase is isolated, repeatedlywashed with water, dried and evaporated under vacuum at 30C. Thecrystallizate obtained on recrystallization from ethyl acetate is washedwith ethyl acetate-l-petroleum ether (2:1) and dried under vacuum atroom temperature, to yield 25.8 g of 2-(p-diphenyl)-isopropyloxycarbonylphenyl ester melting at l 15l 16C with decomposition.

EXAMPLE 6 Eliminating the N-2-(p-diphenyl)-isopropyloxycarbonyl groupfrom different aminoacids N-2-( p-diphenyl )-isopropyloxycarhonylQuantitative aminoacid elimination takes....hoursGlycine-dicyclohexylammonium salt 3% L-leucine 3 L-proline-dicyclohexylammonium salt 3% N -tertiarybutyloxycarbonyl-L-lysinedicyclohexylammonium salt 4%L-tyrosine-cyclohexylammonium salt 2%L-phenylalanine-dicyclohexylammonium salt 3% L-glutamic acid--ytertiarybutylestercyclohexylammonium salt 4 [/5 L-serine-tertiary butylethercyclohexylammonium salt 3 EXAMPLE 7 Eliminating theN-Z-(p-diphenyl)-isopropy1oxycarbonyl group from N-2-(p-diphenyl)-isopropyloxycarbonyl-N -BOC- L-lysyl-N -BOC-L-lysinemethyl ester a. 160.0 mg of N -2-(p-diphenyl)- isopropyloxycarbonyl-N-BOC-L-lysy1-N -BOC-L- lysine methyl ester are dissolved in 2.2 ml of amixture of glacial acetic acid +82.8 percent formic acid+water (7: 1.2parts by volume). After different intervals specimens of 0.3 ml each aretaken, added to 4 ml of dimethylformamide and titrated with0.1N-perchloric acid in glacial acetic acid. After 55 minutes the Nprotective group has been quantitatively eliminated, whereas the N -BOCgroups have remained unaffected.

b. A solution of 240 mg of the above dipeptide derivative in 1.7 ml ofmethylenechloride is mixed at room temperature with 1.3 ml of a solutionof 37.5 g of chloracetic acid in 12.5 ml of water. From the homogeneoussolution specimens of 0.5 ml of each are taken after differentintervals, added to 1.5 ml of methanol and immediately chromatographedon silicagel in the system secondary butanol+glacial acetic acid+water(67:10:23). It is found that after 15 minutes the N protective group hasbeen completely eliminated, whereas the N -BOC groups have remainedunchanged.

The starting material may be prepared thus:

A mixture of 666 mg (1 mmol) of N -2-(pdiphenyl)-isopropyloxycarbonyl-N-BOC-lysine dicyclohexylammonium salt and 4 ml of dimethylformamide ismixed at -15C with 0.125 ml of pivaloylchloride. The whole is stirredfor 15 minutes at C and a solution of 320 mg (1 mmol) of N -BOC-lysinemethyl ester acetate in 3 ml of dimethylformamide is added and the batchis stirred for 2 hours at 0C, then taken up in ethyl acetate, at 0Crepeatedly washed with 0.1 molar citric acid solution, with 2N-sodasolution and with water, dried and evaporated to dryness under vacuum.The residue is dissolved in a little ether, whereupon crystallizationsets in rapidly, to yield 632 mg of N-2-(p-diphenyl)-isopropyloxycarbonyl- N -BOC-lysyl-N -BOC-1ysine methylester melting at 8789C with decomposition. After recrystallization fromethyl acetate+petroleum ether the melting point i is still the same.

EXAMPLE 8 Eliminating the (p-diphenyl)-isopropyloxycarbonyl group from(p-diphenyl)-isopropyloxycarbonyl-L-prolyl-L- leucyl-L-(y-tertiary butylester)-glutamyl-L-phenylalanine-tertiary butyl ester 1.10 g ofN-2-(p-diphenyl)-isopropyloxycarbonyl- Pro-Leu-Glu(OtBU)-Phe-OtBu arestirred for 2% hours with 6.5 ml of a mixture of glacial acetic acid+82.8 percent formic acid+water (7:122 by volume). While cooling withice, the batch is rendered alkaline with saturated potash solution,extracted with ethyl acetate, washed neutral, dried and evaporated todryness under vacuum. The residue is triturated with a small quantity ofpetroleum ether and recrystallized from 75 percent methanol, to yield0.65 g of l-l-Pro-Leu- G1u(OtBu)-Phe-OtBu melting at 188-190C.

The starting material is accessible in the following manner:

5.35 Grams (1O mmols) of N-2-(p-diphenyl)- isopropyloxycarbonyl-prolinedicyclohexylammonium salt in 40 ml of dimethylformamide are mixed at-l5C with 1.25 ml of pivaloylchloride, stirred for minutes at -10C and asolution of 5.8 g of H-Leu-Glu(OtBu)- Phe-OtBu acetate in 30 ml ofdimethylformamide is dropped in. The mixture is allowed to react for 2hours at 0C, then diluted with ethyl acetate and the solution isagitated at 0C twice each with water, 0.1 molar citric acid solution,2N-soda solution and water. The ethyl acetate solution is dried oversodium sulfate and evaporated to dryness; the residue is dissolved inether and caused to crystallize, to yield 6.75 g ofN-2-(pdiphenyl)-isopropyloxycarbonyl-Pro-Leu-Glu(OtBu)- Phe-OtBu which,after recrystallization from 85 percent methanol, melts at 188C withdecomposition.

EXAMPLE 9 Using the N-2-(p-diphenyl)-isopropyloxycarbonyl group in thesolid phase synthesis Manufacturing carbobenzoxy-L-phenylalanyl-L- (N-tertiary butyloxycarbonyl)-lysyl-glycine hydrazide:

2.15 Grams of BOC-glycine resin [styrene-l-divinylbenzene copolymercontaining 0.25 milliequivalent of BOC-glycine/gram resin=0.5 mmol] arestirred for 3 minutes with 10 ml of dioxane, then filtered. Then 10 mlof 4N-hydrochloric acid in dioxane are added and the whole is stirredfor 30 minutes at room temperature and washed with 3 X10 ml of dioxaneand methylenechloride, stirring in each case with the solvent for 3minutes. The batch is then mixed with 10 ml of a mixture ofmethylenechloride+triethylamine (9:1) and stirred for 10 minutes andrinsed with 6X10 ml of methylenechloride. The H-glycine resin is storedunder methylenechloride.

0.83 Gram N -2-(p-diphenyl)- isopropyloxycarbonyl-N -BOC-lysinedicyclohexylammonium salt (1.25 mmols) are dissolved inmethylenechloride and agitated at 0C 3 times each with 0.2 molar citricacid and water. The methylene chloride solution is dried, filtered andconcentrated to a few ml under vacuum. This solution is added to theabove resin, stirred for 5 minutes, then a solution of 290 mg ofdicyclohexylcarbodiimide in a little methylenechloride is added. Thebatch is stirred for 3 hours, filtered and washed with 3X10 ml each ofmethylenechloride, dimethylformamide, dimethylformamide+methanol (1:1)and methylenechloride. The N -2-(pdiphenyl)-isopropyloxycarbonyl-N-BOC-Lys-Gly resin is then suspended in 5 ml of methylenechloride and3.7 ml ofa solution of 37.5 g of chloracetic acid in 12.5 ml of water isadded, the whole stirred on for 1% hours and then washed as describedabove with methylenechloride, dioxane, methylenechloride,methylenechloride+triethylamine (9:1) and methylenechloride. Theresulting l-l-Lys(BOC)-Gly resin is mixed with 0.37 g ofcarbobenzoxy-L-phenylalanine (Z-Phe-Ol-l) in 8 ml of methylenechlorideand stirred for 5 minutes, then 290 mg of dicyclohexylcarbodiimide in alittle methylenechloride is added and the whole is stirred for 3 hours.The Z-Phe-Lys(BOC)-Gly resin is again filtered as described above andwashed with methylenechloride, dimethylformamide,dimethylformamide+methanol (1:1), methanol and ethanol, then stirred for15 hours with 10 ml of ethanol+hydrazine hydrate (3:1 filtered off andrepeatedly washed with ethanol. The combined filtrates are evaporated todryness under vacuum, and the residue is freed from hydrazine hydrate bybeing dried in a high vaccum over concentrated sulfuric acid at 50C. Theresidue is dissolved in 5 m1 of boiling ethanol, a small quantity ofundissolved matter is filtered off and the filtrate is mixed hot withabout 15 ml of water. The batch is seeded, then allowed to cool slowlyto room temperature, and the product which crystallizes out is filteredoff and washed with ethanol+water (1:3), to furnish mg of Z-Phe-Lys(BOC)-Gly-Nl-1Nl-l melting at 163164C.

EXAMPLE 10 Preparation of L-prolyl-L-tyrosyl(N -tertiarybutyloxycarbonyl)-L-lysyl-L-methionine hydrazide with the use of the2-para-diphenylisopropyloxycarbonyl group a. 6.7 (10 mmols) of N2-(para-diphenyl)- isopropyloxycarbonyl-N -BOC-L-lysine-dicyclohexylammonium salt in 40 ml of dimethylformamide are stirred at l 5C whilebeing treated with 1.25 ml of pivaloyl chloride, then stirred at lC forminutes. Under a current of nitrogen, 2.0 g of L-methioninemethylesterhydrochloride and 1.4 ml of triethylamine are then added, and the batchstirred at 0C for 3 hours. It is then diluted with ethyl acetate, washedat 0C 3 times with 0.1-molar citric acid solution, and at roomtemperature three times with 2N-potassium carbonate solution and threetimes with water, then dried and evaporated to dryness under reducedpressure. The crude product (6.5 g) is filtered through a column of 150g of silica gel, and 4.7 g of N -2-(para-diphenyl)-isopropyloxycarbonyl-N -BOC-L-lysyl-L-methionine-methyl ester obtainedin the form of a colorless resin. Rf0.55 in chloroform+acetone (8:2).

b. 3.15 g (5 mmols) of this dipeptide ester are stirred for an hour anda half with 50 ml ofa mixture of glacial acetic acid, 82.8 percentformic acid and water (7:1:2 by volume), then poured into a mixture of50 ml of ethyl acetate and ml of saturated potassium carbonate solutionat 0C. The ethyl acetate solution is washed with water, dried andcarefully evaporated. The resulting N -BOC-L-lysyl-L-methioninemethylester is unstable and must be processed immediately.

c. To this end, the residue is dissolved at 0C in 18 ml of acetonitrileunder nitrogen, treated with a solution of 2.10 g (5 mmols) ofN-2-(para-diphenyl)- isopropyloxycarbonyl-L-tyrosin (liberated from thecyclohexylammonium salt by means of citric acid) in 2 ml ofdimethylformamide and 1.15 g of dicyclohexylcarbodiimide, and stirred at0C for 20 hours. The dicyclohexylurea which precipitates is filteredoff, the filtrate evaporated, the residue triturated withether+petroleum ether, filtered off, and reprecipitated from ethylacetate+petroleum ether. There are obtained 2.8 g ofN-2-(para-diphenyl)-isopropyloxycarbonyl-L- tyrosyl-N'-BOC-L-lysyl-L-methionine-methylester in the form of a powder which isunitary according to thin layer chromatography; RFOQ inchloroform+acetone (8:2).

d. 2.38 g (3 mmols) of this tripeptide ester are stirred with 30 ml of amixture of glacial acetic acid 82.8 percent formic acid+water (711:2 byvolume) for 1 hour at room temperature. The solution is poured into icewater, extracted twice with ether, the aqueous solution renderedalkaline with saturated potassium carbonate solution, and extracted withethyl acetate. On evaporation of the ethyl acetate there remain 1.33 gof L-tyrosyl-N -BOC-L-lysyl-L-methioninemethyl ester in the form of acolorless foam. Rf=0.5 in chloroform- +methanol (85:15).

e. 1.1 l g (2 mmols) of this tripeptide ester and 0.71 g (2 mmols) ofN-2-(para-diphenyl)- isopropyloxycarbonyl-L-proline (liberated from thedicyclohexylammonium salt) are dissolved in 6 ml of acetonitrile, thesolution treated at 0C with 450 mg of dicyclohexylcarbodiimide, andstirred at 0C for 18 hours. The dicyclohexylurea which precipitates isfiltered off, the filtrate diluted with ethyl acetate, extracted byagitation at 0C three times with 0.1-molar citric acid, at roomtemperature with 2N-potassium carbonate solution and water, then dried,and evaporated to dryness under reduced pressure.N-2-(paradiphenyl)-isopropyloxycarbonyl-L-prolyl-L-tyrosyl- N-BOC-Llysyl-L-methionine-methyl ester is obtained in quantitative yield(1.8 g).

f. The methyl ester is stirred for one hour with 22 ml of a7:1:2-mixture of glacial acetic acid, 82.8 percent formic acid andwater. The solution is treated with iced water, extracted twice withether, the aqueous solution is given an alkaline reaction with saturatedpotassium carbonate solution, and extracted with ethyl acetate. Afterdrying, the ethyl acetate is completely expelled under reduced pressure.There remain behind 1.02 g of L-prolyl-L-tyrosyl-N-BOC-L-lysyl-L-methioninemethyl ester; Rf=0.25 in chloroform+methanol(:15). The product can be crystallized from acetonitrile. Melting point,l77l80C.

g. 326 mg (0.5 mmol) of the tetrapeptide ester are dissolved in 1 ml ofhot methanol, treated at room temperature with 0.25 ml of hydrazinehydrate while nitrogen passes over, then kept under seal for 18 hours.The product which crystallizes is filtered off and washed with water.225 mg of L-prolyl-L-tyrosyl-N -BOC-L- lysyl-L-methionine hydrazide ofmelting point 206207C are obtained.

EXAMPLE 1 1 Preparation of L-prolyl-L-tyrosyl-l l -tertiarybutyloxycarbonyl-L-lysyl-L-methionine hydrazide by the solid phasesynthesis with the use of the 2-(para-diphenyl)-isopropyloxycarbonylgroup a. In a nitrogen atmosphere in a reaction vessel of 50 ml capacityaccording to Merrifield, 4.9 g of BOC- methionine resin(styrol-divinylbenzene-copolymer containing 0.18 m equivalentBOC-methionine/g resin=0.88 mmol) are agitated in turn with thefollowing reagents which are each filtered off before the next is used:

. 25 ml of dioxan for 4 minutes;

. 20 ml of 4N-HC1 in dioxan for 60 minutes;

. 3X20 ml of dioxan for 3 minutes each time;

. 3X20 m1 of ethanol for 3 minutes each time;

. 3X20 ml of chloroform for 3 minutes each time; 20 ml ofchloroform+triethylamine (9:1) for 10 minutes;

7. 3X20 ml of chloroform for 3 minutes each time;

8. 4X20 ml of methylene chloride for 3 minutes each time.

The resulting methionine resin is stored moist with methylene chloride(it contains a total of 17 ml of methylene chloride). From 1.87 g of N-2-(paradiphenyl)-isopropyloxycarbonyl-N -tertiarybutyloxycarbonyl-L-lysine-dicyclohexyl-ammonium salt the protectedL-lysine is liberated as described in Example 9. The resulting methylenechloride solution is added to the aforementioned resin, the wholeagitated for 10 minutes, then a solution of 730 mg ofdicyclohexylcarbodiimide in 3 ml of methylene chloride is added, and thewhole agitated for 4 hours. The resin is then washed while beingagitated with 3X20 ml of methylene chloride (3 minutes each time);

3X20 ml of methanol (3 minutes each time); and

X20 ml of methylene chloride (3 minutes each time). The resulting N-2-(para-diphenyl)- isopropyloxycarbonyl-N -BOC-L-lysyl-L-methionineresin (which contains 17 ml of methylene chloride) is treated with 12.6ml of a 75 percent aqueous chloracetic acid solution and agitated for anhour and a half to eliminate the N-2-(para-diphenyl)-isopropyloxycarbonyl group. The resin is filtered offand washed while being agitated with:

3X20 ml of methylene chloride (3 minutes each time);

4X20 ml of ethanol (3 minutes each time);

25 ml of chloroform (3 minutes);

20 ml of chloroform+triethylamine (9:1) (10 minutes);

3X20 ml of chloroform (3 minutes each time);

3X20 ml of methanol (3 minutes each time); and

20 ml of methylene chloride (3 minutes each time).

In a manner analogous to that described above, the resulting N-BOC-L-lysyl-L-methionine resin is reacted withN-Z-(para-diphenyl)-isopropyloxycarbonyl- L-tyrosine. To this end, 1.6 gof the cyclohexylammonium salt of this compound are stirred at C with 25ml of ethyl acetate and 20 ml ofa l-molar citric acid solution untildissolution is complete. The aqueous layer is separated, the ethylacetate solution is washed, dried, and evaporated to dryness in vacuo.The crystalline residue is dissolved in 1 ml of dimethyl formamide,diluted with 10 ml of methylene chloride, and the solution added to theaforementioned resin. At the same time, 710 mg of hydroxysuccinimide areadded. The

batch is agitated for 10 minutes, and a solution of 700 mg ofdicyclohexylcarbodiimide in ml of methylene chloride then added, and thewhole is agitate for 4 hours. The resin is then washed as describedabove with methylene chloride, methanol and methylene chloride. For theelimination of the N-2-(para-diphenyl)-isopropyloxycarbonyl group withchloracetic acid the above procedure is followed exactly. L-Tyrosyl-NBOC-L-lysyl-L-methionine resin is obtained which is condensed withN-2-(para-diphenyl)- isopropyloxycarbonyl-L-proline in methylenechloride (obtained from a solution of 1.34 g of the dicyclohexylammoniumsalt in methylenechloride by liberation with citric acid). The resultingmethylene chloride solution is agitated with the resin for minutes, asolution of 580 mg of dicyclohexylcarbodiimide in 5 ml of methylenechloride is added, and the whole agitated for 18 hours. The resin isfiltered off, washed as aforedescribed, and theN-Z-(para-diphenyl)-isopropyloxycarbonyl group split off withchloracetic acid. After the usual washing procedures, the resultingL-propyl-L- tyrosyl-N -BOC-L-lysyl-L-methionine resin is agitated with20 ml of ethanol, filtered and, under a weak current of nitrogen,stirred with 7 ml of ethanol and 3 ml of hydrazine hydrate for 17 hours.The resin is filtered off and washed ten times with 10 ml of 95 percentethanol each time. The combined filtrates are evaporated under reducedpressure at 50C and the residue dried in a high vacuum over sulfuricacid. The crude product is dissolved in ml of 0. lN-acetic acid and 80ml of ethyl acetate, and the aqueous solution agitated with 2X50 ml ofethyl acetate. The three ethyl acetate solutions are extracted twicewith 10 ml each time of 0.1-normal acetic acid. The three aqueousextracts are combined and lyophilized. Further purification is performedby chromatography over 7 g of CM-Sephadex (Registered Trade Mark) bymeans of a gradient consisting of 300 ml of 0.01-molar ammoniumacetate-and 300 ml of 0.05 molar ammonium acetate pH 6.5. The purefractions are combined and lyophilized. 320 mg of the acetate ofL-prolyl-L-tyrosyl-N -BOC-L-lysy1-L- methionine hydrazide are obtained.The product is fully identical with the product described in Example 10.Rf=0.35 in the thin-layer chromatogram, system s-butanol+glacial aceticacid+water (67:10:23). The basic product is liberated from the acetateby means of potassium carbonate solution and then crystallizes fromaqueous methanol. Melting point 206207C (with decomposition).

EXAMPLE 12 Preparation ofN-2-(para-diphenyl)-isopropy1oxycarbonyl-Lleucyl-L-valyl-S-trityl-L-cysteinyl-glycyl-L-glutamic acid-di-tertiary butylester.

The starting material, S-trityl-L-cysteinyl-glycyl-L- glutamicacid-di-tertiary butylester, can be prepared as follows: Fromcarbobenzoxy-glycine and L-glutamic acid-di-tertiary butyl ester,carbobenzoxyglycyl-L- glutamic acid-di-tertiary butylester is obtainedby the mixed anhydride method. Melting point, 74-76C. Theglycyl-L-glutamic acid-di-tertiary butylester obtained therefrom byhydrogenation is reacted with N,S-di-trityl-L-cystein anddicyclohexylcarbodiimide and yieldsN,S-Di-trityl-L-cysteinyl-glycyl-L-glutamic acid-di-tertiary butylestermelting at 98110C. On elimination of the N-trityl group with percentacetic acid, S-trityl-L-cysteinyl-glycyl-L-glutamic acid-ditertiarybutyl ester is obtained.

N-Z-(para-diphenyl)-isopropyloxycarbonyl-L-valin (liberated from 1.91 g(4.2 mmols) of the cyclohexylammonium salt by means of citric acid) isdissolved in 20 ml of ethyl acetate, and the solution treated with 0.59ml (4.2 mmols) of triethylamine and, after cooling to 10C, with 0.56 ml(4.2 mmols) of chlorocarbonic acid-isobutyl ester. The batch is stirredfor 10 minutes at l0C before a solution of 2.78 g (4.2 mmols) of theabove tripeptide-diester in 25 m1 of ethyl acetate is added dropwise,and then stirred on for 15 minutes at 10C and 1 hour at roomtemperature. After dilution with ethyl acetate, the batch is extractedby agitation with 0.5-mo1ar citric acid, Nsodium bicarbonate, and waterat 0C, dried, and evaporated to dryness. The residue is crystallizedfrom acetone+ether to obtain 2.91 g of N-2-( paradiphenyl)-isopropyloxycarbonyl-L-valyl- S-trityl-L-cysteinyl-glycyl-L-glutamicacid-di-tertiary butyl ester melting at l84-186C. Rf=0.6 inchloroform+methanol (:5).

1 g (1 mmol) of this tetrapeptide ester is stirred at room temperaturefor 1 hour with 10 m1 of a mixture of glacial acetic acid, 82.8 percentformic acid, and water (7: 1:2 by volume). After the addition of 10 mlof water and 40 ml of chloroform, the batch is given an alkalinereaction with concentrated ammonia at 0C, the chloroformic solutionseparated, washed, dried and evaporated to dryness. The residue istriturated with petroleum ether to obtain 0.76 g of L-valyLS-trityl-L-cysteinyl-glycy1-L-glutamic acid-di-tertiary butyl ester in the form ofan oil which is unitary according to thinlayer chromatography. Rf=0.25in chloroform+methanol (95:5). At 0C, 238 mg of dicyclohexylcarbodiimideare added to 370 mg (1 mmol) of N2- (para-diphenyl)-isopropyloxycarbonyl-L-leucine and 768 mg of the above tetrapeptide ester in 7 ml oftetrahydrofuran, and the whole is stirred at C for 4 hours. Filteringoff the dicyclohexylurea is followed by dilution with ethyl acetate,washing at 0C with 0.5 molar citric acid, N-sodiumbicarbonate and water,drying, and evaporation. By elution with ethyl acetate in thechromatography on silica gel, 775 mg ofN-Z-(paradiphenyl)-isopropyloxycarbonyl-L-leucyl-L-valyl-S-trityl-L-cysteinyl-glycyl-L-glutamic acid-di-tertiary butylester areobtained as a unitary product. Rf=0.55 in chloroform+methanol (9:1);Rf0.63 in tolueneacetone (1:1).

EXAMPLE l3 lntroduction of the 1,1-diphenyl-ethoxycarbonyl group 8.2 gof diphenyl-methyl-carbinol, 5.35 g of isocyanate-acetic acid ethylester and 4 ml of pyridine are heated at 50C for 48 hours. The solutionis diluted with 100 ml of ether, extracted at 0C with 0.1-molar citricacid solution and water, dried over sodium sulfate, and evaporatedcompletely under reduced pressure. There are obtained 10.6 g of viscousoil consisting of 1,l-diphenyl-ethoxycarbonyl-glycine-ethyl ester.Rf=0.7 in the thin-layer chromatography in chloroform+methanol (95:5).

EXAMPLE 14 Splitting off the 1,1-diphenyl-ethoxy-carbonyl'group-diphenyl-propyloxycarbonyl 207.8 mg of 1,1-diphenylethoxycarbonylglycineethyl ester are dissolved at 25C in 3.15ml of 80 percent acetic acid. At different times, test portions of 0.20ml are taken, added to 4 ml of dimethylformamide, and the liberatedglycine ethyl ester titrated with 0.1N-perchloric acid. The half-lifeperiod for the elimination of the protective group is about 30 minutes.After hours the elimination is quantitative.

EXAMPLE 15 EXAMPLE 16 Elimination of the l,1-diphenyl-propyloxycarbonylgroup 409 mg of 1,1-diphenyl-propyloxycarbonyl-glycineethyl ester aredissolved at 22C in 80 percent acetic acid. At different times testportions of 0.5 ml are taken, added to 4 ml of dimethylformamide, andthe liberated glycine ethylester titrated with 0.1N- perchloric acid.The half-life period for the elimination of the protective group isabout 50 minutes. After 8 hours the protective group is split offquantitatively.

group and then eliminating the acyl group introduced,.

wherein the protective group used is the group of the formula 1 in whichR, represents lower alkyl, R, lower alkyl or phenyl, and R stands forphenyl, and wherein the phenyl radicals are unsubstituted or substitutedby lower alkyl, phenyl or lower alkylphenyl groups.

2. Process according to claim 1, wherein a group of the formula I isused in which R, and R each represents a methyl or ethyl group and R,has the same meaning as in claim 1.

3. Process according to claim 1, wherein a group of the formula I isused in which R represents a phenyl, tolyl or biphenylyl group and R,and R stand for lower alkyl.

4. Process according to claim 1, wherein R, stands for methyl or ethyl,and R and R each stands for phenyl.

5. Process according to claim 1, wherein the protective group of theformula 1 is the Z-phenyl-isopropyloxycarbonyl group or the1,1-diphenylethyloxycarbonyl group.

6. Process according to claim 1, wherein the protective group of theformula 1 is the 2-para-tolylisopropyloxycarbonyl group.

7. Process according to claim 1, wherein the protective group of theformula I is the 2-(para-biphenylyl)- isopropyloxycarbonyl group.

8. Process according to claim 1, wherein the group of the formula 1 isused as a selectively eliminable a-amino group in addition toacid-labile side-chain protective groups.

9. Process according to claim 1, wherein in the synthesis of peptideswhich in the side-chain contain amino, hydroxy or carboxyl groups, theamino groups are protected by the tertiary butyloxycarbonyl group, thehydroxy group by the tert. butyl ether group and the carboxyl group bythe tertiary butylester group and the a-amino group by the group of theformula 1.

10. Process according to claim 1, wherein, in the synthesis of peptides,containing mercapto groups in the side-chain, these groups are protectedby the trityl group and the oz-amino group by the group of the formulaI.

11. Process according to claim 1, wherein the group of the formula I isused in the synthesis of peptides by the solid phase synthesis.

12. Process according to claim 1, wherein the group of the formula I isintroduced by means of its azide into a-amino acids or into peptidesconsisting of a-amino acids.

13. Process according to claim 1, wherein the group of the formula 1 isintroduced by means of its phenyl ester into a-amino acids or intopeptides consisting of a-amino acids.

14. N -Acyl-a-aminoacids and derivatives thereof in which the acylradical is a group of the formula I -oco- (I) in which R represents alower alkyl radical, R stands for lower alkyl or phenyl, and R forphenyl, and wherein the phenyl radicals are unsubstituted or substitutedby lower alkyl, phenyl or lower alkylphenyl groups.

15. N -Acyl-a-aminoacids and derivatives thereof in which the acylradical is a group of the formula I as claimed in claim 14 in which R,and R each represents methyl or ethyl and R stands for phenyl, tolyl orbiphenylyl.

16. N -Acyl-a-aminoacids and derivatives thereof, in which the acylradical is a group of the formula I as calimed in claim 14 in which Rand R each represents methyl and R stands for a phenyl group.

17. N -Acyl-a-aminoacids and derivatives thereof, in which the acylradical is a group of the formula I as claimed in claim 14 in which Rand R each represents methyl and R stands for para-biphenylyl.

18. N -Acyl-a-aminoacids and derivatives thereof in which the acylradical is a group of the formula I as claimed in claim 14 in which R,and R each represents a methyl group, and R stands for para-tolyl.

1. PROCESS FOR THE TEMPORARY PROTECTION OF AMINO GROUPS IN PEPTIDESYNTHESES BY ACYLATING THE AMINO GROUP AND THEN ELIMINATING THE ACYLGROUP INTRODUCED, WHEREIN THE PROTECTIVE GROUP USED IS THE GROUP OF THEFORMULA I
 2. Process according to claim 1, wherein a group of theformula I is used in which R1 and R2 each represents a methyl or ethylgroup and R3 has the same meaning as in claim
 1. 3. Process according toclaim 1, wherein a group of the formula I is used in which R3 representsa phenyl, tolyl or biphenylyl group and R1 and R2 stand for lower alkyl.4. Process according to claim 1, wherein R1 stands for methyl or ethyl,and R2 and R3 each stands for phenyl.
 5. Process according to claim 1,wherein the protective group of the formula I is the2-phenyl-isopropyloxycarbonyl group or the 1,1-diphenylethyloxycarbonylgroup.
 6. Process according to claim 1, wherein the protective group ofthe formula I is the 2-para-tolylisopropyloxycarbonyl group.
 7. Processaccording to claim 1, wherein the protective group of the formula I isthe 2-(para-biphenylyl)-isopropyloxycarbonyl group.
 8. Process accordingto claim 1, wherein the group of the formula I is used as a selectivelyeliminable Alpha -amino group in addition to acid-labile side-chainprotective groups.
 9. Process according to claim 1, wherein in thesynthesis of peptides which in the side-chain contAin amino, hydroxy orcarboxyl groups, the amino groups are protected by the tertiarybutyloxycarbonyl group, the hydroxy group by the tert. butyl ether groupand the carboxyl group by the tertiary butylester group and the Alpha-amino group by the group of the formula I.
 10. Process according toclaim 1, wherein, in the synthesis of peptides, containing mercaptogroups in the side-chain, these groups are protected by the trityl groupand the Alpha -amino group by the group of the formula I.
 11. Processaccording to claim 1, wherein the group of the formula I is used in thesynthesis of peptides by the solid phase synthesis.
 12. Processaccording to claim 1, wherein the group of the formula I is introducedby means of its azide into Alpha -amino acids or into peptidesconsisting of Alpha -amino acids.
 13. Process according to claim 1,wherein the group of the formula I is introduced by means of its phenylester into Alpha -amino acids or into peptides consisting of Alpha-amino acids.
 14. N -Acyl- Alpha -aminoacids and derivatives thereof inwhich the acyl radical is a group of the formula I
 15. N -Acyl- Alpha-aminoacids and derivatives thereof in which the acyl radical is a groupof the formula I as claimed in claim 14 in which R1 and R2 eachrepresents methyl or ethyl and R3 stands for phenyl, tolyl orbiphenylyl.
 16. N -Acyl- Alpha -aminoacids and derivatives thereof, inwhich the acyl radical is a group of the formula I as calimed in claim14 in which R1 and R2 each represents methyl and R3 stands for a phenylgroup.
 17. N -Acyl- Alpha -aminoacids and derivatives thereof, in whichthe acyl radical is a group of the formula I as claimed in claim 14 inwhich R1 and R2 each represents methyl and R3 stands forpara-biphenylyl.
 18. N -Acyl- Alpha -aminoacids and derivatives thereofin which the acyl radical is a group of the formula I as claimed inclaim 14 in which R1 and R2 each represents a methyl group, and R3stands for para-tolyl.